The present invention relates to a method of identifying and quantifying specific fungi and bacteria using specific DNA sequences, as described and taught herein. These sequences can be used with real time detection of PCR products with a fluorogenic probe system or other molecular approaches like hybridizations.
Fungi and bacteria are the source of or contribute to many health problems including infections, gastroenteritis, ulcers, asthma, allergies and sinusitis. The rapid identification of these microorganisms is critical for diagnosis and treatment. In addition, detecting and/or quantifying these microorganism in the environment may help to prevent adverse health effects.
Limitations of Current Technology
In order to determine the risk fungi and bacteria pose to human health, it is necessary to know what fungi and bacteria are present and in what numbers. Fungi and bacteria can be ingested, inhaled, or might enter the body through abrasions or punctures. It is important to identify these microorganisms as specifically and as rapidly as possible. Some species of a particular genus are harmless whereas others of the same genus may cause significant health effects. So without knowing precisely what microorganisms are present and in what numbers, it is impossible to evaluate the potential for negative health effects or the establishment of a risk assessment.
In the past, the detection and quantitative measurement of fungi and bacteria in samples has been performed either by direct microscopic examination of the collected cells or by growing cells on a suitable medium and identification and enumeration of the resultant colonies. The first method is highly labor intensive and is subject to potential errors in the recognition and positive identification. The second method is both time consuming and subject to significant quantitative inaccuracy. Both methods require extensive experience on the part of the analyst.
Some molecular approaches, such as the conventional polymerase chain reaction (PCR) procedure, are subject to inaccuracies due to the difficulty of quantifying the product. This procedure is also relatively slow and requires expertise in molecular biology.
It is an object of the present invention to overcome the aforesaid deficiencies in the prior art.
It is an object of the present invention to provide a simple, reliable method for detecting and quantifying some fungi and bacteria by using the nucleotide sequences specific to each species or group of species of fungi and bacteria, as described herein.
According to the present invention, fungi and bacteria can be identified and quantified by using a nucleotide sequence specific to the particular species or, in the case of some fungi, group of species. Many methods including using real time, probe-based detection of polymerase chain reaction (PCR) products (e.g. TaqMan(trademark) system) or other methods of detection and quantification including hybridization or conventional PCR could be used with these sequences.
Theory
Each microorganism is unique because of the sequence of some of the nucleotides in its DNA. However, there are many sequences which are common to more than one organism. There is thus a hierarchy or classification into which all microorganisms can be arranged. The xe2x80x9cspeciesxe2x80x9d are typically the finest level of distinction that is recognized for separation of different members of a given genus. In the past, species were separated on the basis of morphological or biochemical differences. In order to identify or separate different species on the basis of its DNA sequence, one finds sequences that are unique to a given species but at the same time common to all isolates of a given species.
For this invention the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (rDNA) of the different fungi were used. For the bacteria, the sequences of unique enzymes were chosen.
To apply this invention, a number of possible detection methods are possible. For example, the TaqMan(trademark), 3xe2x80x2-5xe2x80x2 exonuclease assay signals the formation of PCR amplicons by a process involving the nucleolytic degradation of a double-labeled fluorogenic probe that hybridizes to the target template at a site between the two primer recognition sequences (cf. U.S. Pat. No. 5,876,930). The model 7700 automates the detection and quantitative measurement of these signals, which are stoichiometrically related to the quantities of amplicons produced, during each cycle of amplification In addition to providing substantial reductions in the time and labor requirements for PCR analyses, this technology permits simplified and potentially highly accurate quantification of target sequences in the reactions.
There are additional systems and other molecular approaches that operate upon essentially the same principal. What is common to all of these technologies is the need for the identification of specific sequences that are unique to the targeted organism but common to all members of the species. The present invention teaches these identifying sequences and gives a description of the practical application of the sequences in the identification and quantification of specific fungi and bacteria.